Preparation method of greater omentum acellular matrix and construction method of cartilage tissue

A technology of decellularized matrix, construction method, applied in the field of tissue engineering

Pending Publication Date: 2020-05-08
GUANGDONG HONGZHI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on the above-mentioned present situation, the main purpose of the present invention is to provide a preparation method of omentum acellular matrix

Method used

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  • Preparation method of greater omentum acellular matrix and construction method of cartilage tissue
  • Preparation method of greater omentum acellular matrix and construction method of cartilage tissue
  • Preparation method of greater omentum acellular matrix and construction method of cartilage tissue

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preparation example Construction

[0097] Due to the characteristics of cartilage tissue, the omentum acellular matrix material prepared by the existing method is not suitable for the regeneration of cartilage tissue. Based on this, the application provides a preparation method of the omentum acellular matrix material. Of course It can be understood that the omentum acellular matrix material prepared by the preparation method provided in this application is especially suitable for the regeneration culture of cartilage tissue, and of course, it can also be applied to the regeneration culture of other tissues. like figure 1 Shown, this preparation method comprises steps:

[0098] S100, cleaning the omentum;

[0099]S200, performing freeze-thaw cycles on the washed omentum to obtain a first intermediate;

[0100] S300, adding the first intermediate to a solution containing trypsin and phenylmethylsulfonyl fluoride, with a pH value of 7.8 to 8.2, and continuing for a first predetermined period of time to obtain a...

Embodiment 1

[0107] Fresh porcine omentum was washed 3 times with normal saline;

[0108] Add the washed omentum to a pH containing 10mmol / L trishydroxymethylaminomethane (Tris), 10mmol / L ethylenediaminetetraacetic acid (EDTA) and 1% by mass percent phenylmethylsulfonyl fluoride (PMSF). 3 freeze-thaw cycles in the buffer solution of =8.0, wherein, in one freeze-thaw cycle, the freezing temperature is -80°C, the duration is 1 hour, and the melting temperature is 37°C, and the duration is 0.5 hour;

[0109] The tissue after freeze-thaw cycle treatment was added to a solution containing 0.25% trypsin by mass percentage, 0.1% ethylenediaminetetraacetic acid (EDTA) by mass percentage, and 1% phenylmethylsulfonyl fluoride (PMSF) by mass percentage. Shake at 37°C for 12 hours;

[0110] The tissues obtained in the previous steps were added to isopropanol and degreased by shaking at 37°C for 48 hours, and then phosphate-buffered saline PBS (8g / L NaCl, 200mg / L KCl, 1g / L NaCl, pH=8.0 2 HPO 4 , 200...

Embodiment 2

[0115] Fresh porcine omentum was washed 3 times with normal saline;

[0116] Add the washed omentum to a pH containing 1mmol / L trishydroxymethylaminomethane (Tris), 1mmol / L ethylenediaminetetraacetic acid (EDTA) and 0.1% by mass phenylmethylsulfonyl fluoride (PMSF). 3 freeze-thaw cycles in the buffer solution of =7.8, wherein, in one freeze-thaw cycle, the freezing temperature is -20°C and the duration is 8 hours, and the melting temperature is 30°C and the duration is 1 hour;

[0117] The tissue after freeze-thaw cycle treatment was added to a solution containing 0.1% trypsin by mass percentage, 0.01% ethylenediaminetetraacetic acid (EDTA) by mass percentage, and 0.1% phenylmethylsulfonyl fluoride (PMSF) by mass percentage. Shake at 30°C for 6 hours;

[0118] The tissues obtained in the previous steps were added to isopropanol and degreased by shaking at 30°C for 40 hours, and then phosphate-buffered saline PBS (8g / L NaCl, 200mg / L KCl, 1g / L NaCl, pH=7.8 2 HPO 4 , 200mg / L K...

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Abstract

The invention provides a preparation method of a greater omentum acellular matrix and a construction method of cartilage tissue. The preparation method comprises the following steps: cleaning a greater omentum, performing freezing and thawing cycle treatment so as to obtain a first intermediate, adding the first intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluorideand has a pH value of 7.8-8.2, maintaining the intermediate in the solution for a first preset period of time so as to obtain a second intermediate, performing degreasing treatment on the second intermediate, performing cleaning so as to obtain a third intermediate, adding the third intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluoride and has a pH value of 7.8-8.2,maintaining the intermediate in the solution for a second preset period of time so as to obtain a fourth intermediate, performing tissue cell removal treatment on the fourth intermediate, performingcleaning so as to obtain a fifth intermediate, and performing drying and sterilization treatment on the fifth intermediate, so as to obtain a greater omentum acellular matrix material. The greater omentum acellular matrix prepared by using the preparation method provided by the invention is particularly applicable to regeneration culture of the cartilage tissue.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a method for preparing omentum acellular matrix material and a method for constructing cartilage tissue. Background technique [0002] Trauma, osteoarthritis, etc. are common orthopedic diseases in clinical practice. The incidence of this disease is relatively high, and there are relatively many incentives, and most patients are accompanied by subchondral bone defects after injury. The repair of the joint will induce other diseases, and even lead to the loss of joint function in severe cases. At present, there is still no ideal repair method for osteoarticular cartilage defects clinically, and the commonly used repair methods still have certain limitations. The traditional cartilage injury treatment methods include arthroplasty, drilling, microfracture and arthroscopic lavage. However, none of these methods can restore the damaged cartilage and subchondral bone to the...

Claims

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Application Information

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IPC IPC(8): A61L27/36C12N5/077
CPCA61L27/3604A61L27/3654A61L27/3687A61L27/3691A61L2430/06A61L2430/40C12N5/0655C12N2533/92
Inventor 万绵水林创鑫
Owner GUANGDONG HONGZHI BIOTECHNOLOGY CO LTD
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